Many important biological determinants of HIV-1 disease progression and transmission map to the envelope glycoprotein, including antibody neutralization susceptibility, viral tropism for entry and epitopes for T cell response. Genetic and phenotypic characterizations of the envelope gene suggest a bottleneck effect in the spread of the virus to other body compartments and transmission, resulting in a predominantly homogenous viral population. However, the limitations in current methods to accurately assign HIV-1 coreceptor usage, distinguish genetic variations and detect minor variants in the HIV quasispecies within a patient and between transmission pairs have led to controversial findings. Furthermore, the small patient sample size studied has been too small to make broad generalization regarding mechanisms of HIV transmission and pathogensis. Lastly, these various properties of the envelope gene have been studied predominantly in subtype B infection and less clearly elucidated in HIV-1 subtype C, the most rapidly spreading and prevalent infection worldwide. We hypothesize that a heterogenous HIV-1 population with different envelope sequences and coreceptor usage are transmitted during infection but is not fully characterized and measured by our current available techniques. We propose to use novel and more sensitive methods to determine coreceptor usage and to perform in-depth sequencing and genetic analyses of the envelope gene from a large subtype C clinical cohort from Botswana. The envelope gene in subtype C infection will be studied in longitudinal samples, as well as between plasma-breast milk and mother-infant pairs. We plan to study in greater depth the properties of the HIV-1 subtype C envelope glycoprotein with the following specific aims: 1) identify clinical and genetic determinants of coreceptor switching, 2) assess for correlates of breastfeeding transmission of HIV-1 and 3) examine pattern of viral transmission among mother-to-child transmitting pairs occurring through different routes of MTCT using ultradeep sequencing. The results of this study will further the understanding of viral evolution in the context of transmission and disease progression, identify clinical correlates of transmission and disease progression, and ultimately give insights into potential strategies of prevention, treatment and vaccine development in those parts of the world most affected by HIV. PUBLIC HEALTH RELEVANCE (provided by applicant): Understanding mechanisms of HIV pathogenesis, transmission and clinical progression will have public health relevance by guiding the design of HIV prevention and treatment strategies.